Breast cyst fluid protein assay

ABSTRACT

This disclosure relates to assay of a glycoprotein component of human breast gross cystic disease fluid which has been designated GCDFP-15. This material is a useful marker in monitoring the efficacy of therapy in women with metastatic breast carcinoma and also in determining the maturity of the fetus in pregnant women. The assay for GCDEP-15 can also be used in conjunction with other assays for breast carcinoma such as an assay for carcinoembryonic antigen (CEA) whereby the utilization of both tests is more effective in monitoring for recurrence of disease than using either assay alone.

This application is a continuation-in-part of Ser. No. 102,095, filedDec. 10, 1979, which is a divisional of Ser. No. 880,257, filed Feb. 22,1978, now U.S. Pat. No. 4,229,426.

BACKGROUND OF THE INVENTION

Human breast gross cystic disease has been investigated by C. D.Haagensen, Diseases of the Breast, Second Edition, Chapter 7, CysticDisease of the Breast (W. B. Saunders, Philadelphia, 1971). It has beenfound that patients who develop this disease have approximately afour-fold increased incidence of breast carcinoma above normal. Thisdata suggests a significant link between breast carcinoma and grosscystic disease.

Up to the present, biochemical analysis of breast cystic disease fluidhas been limited and incomplete. High levels of CEA have been found inthe fluid as well as high potassium levels indicating anintracellular-like ionic consistency. See, in this regard, two papers byJ. Fleisher and coworkers:

Clin. Chem., 20/1, 41 (1974); and

Memorial Sloan-Kettering Cancer Center Clinical Bulletin, pages 94-97(1974).

A report of clinical evaluation of the GCDFP-15 immunoassay of thepresent invention has been published in Ann. Surg., 185, 279 (1977).Methods for diagnosing, identifying and detecting carcinoembryonicantigen are disclosed in U.S. Pat. No. 3,663,684. An assay for the A andB components of carcinoembryonic antigen is disclosed in U.S. Pat. No.3,697,638.

DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that a specificglycoprotein isolated from human gross cystic disease fluid can serve asa marker detectable in the systemic circulation of patients with breastcarcinoma. Such glycoprotein can be conveniently detected by immunoassaytechniques particularly by radioimmunoassay.

Gross cystic disease fluid appears to be a unique secretion from breastepithelial cells, probably in response to abnormal hormonal stimulation.Of significant importance is the relative uniformity of gross cysticdisease fluid proteins (GCDFP) as judged by sodium dodecyl sulfate (SDS)and acrylamide gel analysis from patient to patient indicating a commonpathophysiological etiology. Four major component proteins appearpresent in all GCDFP samples. Such proteins are designated GCDFP-70,GCDFP-44, GCDFP-24 and GCDFP-15.

The GCDFP-70 component has been found to be immunologically identical tohuman albumin. It is present in trace amounts in GCDFP relative toplasma, approximately 1/100th the plasma concentration.

The GCDFP-44 component has been found to be immunologically identical toplasma Zn-alpha₂ -glycoprotein. The GCDFP-44 component is found at anapproximately fifty-fold higher concentration in GCD fluid than inplasma.

The GCDFP-24 component appears to be a progesterone binding glycoproteinand is immunologically identical to an unidentified human plasmacomponent which is present in Cohn Fraction VI. The GCDFP-24 componentis at approximately a 100-fold higher concentration in GCD fluid (10-50mg/ml) than the immunologically cross identical component of humanplasma. This GCDFP-24 component has been described by Pearlman andco-workers, cf. J. Biol. Chem. 248/16, 5736 (1973); J. Endocrinology75/3, p. 19 (1977).

Finally, unlike the other major glycoprotein components of GCD fluid,the GCDFP-15 component is not immunologically identical to anycomponents of plasma as determined by Ouchterlony analysis. However,GCDFP-15 was found to have immunological cross-identity with a componentpresent in both human milk and human saliva. The GCDFP-15 component thusrepresents a specific epithelial cell secretion. Therefore this GCDFP-15component is particularly suitable to serve as a plasma marker forbreast carcinoma monitoring due to carcinoma cell secretions into thesystemic circulation.

The isolation of GCDFP-15 from human gross cystic disease fluids can beaccomplished by utilizing conventional protein isolation techniques.Thus, GCD fluid samples after ultracentrifugation may be chromatographedon a column of Sephadex G-200 which results in two main elution peakscorresponding to molecular sizes of 140,000 (Pool I) and 70,000 (PoolII). The Sephadex G-200 Pool II material is then further fractionated byHydroxylapatite column chromatography and DEAE-Agarose step gradient.Purification of the GCDFP-15 component is achieved by recycling thedesired eluate through the DEAE-Agarose step gradient.

More specifically, the purification of GCDFP-15 can be carried out asfollows. Breast GCD fluid was obtained by needle aspiration from womenunder treatment for gross cystic disease. The GCD fluid specimens werestored at -10° C. Initial processing of specimens consisted ofultracentrifugation at 160,000×G for one hour.

Fifteen separate GCD fluid samples were individually columnchromatographed on Sephadex G-200 (Pharmacia) in a 2.6×80 cm column at10° C. Ammonium acetate buffer, 0.1 M, pH 6.75, was used as the eluentwith an elution rate of 16 ml/hr by gravity flow. Absorbance of elutedfractions was determined at 280 nm. Each sample gave a slightlydifferent elution profile. Several of the samples demonstrated twoseparate peaks of approximately 140,000 and 70,000 molecular size. Thefifteen individual Sephadex G-200 separated samples were pooled intoregions I and II approximating these two peaks.

The Sephadex G-200 region II pool was further fractionated byHydroxylapatite chromatography using Hydroxylapatite (Biorad) in a1.5×30 cm column. The Hydroxylapatite was pre-equilibrated in 0.01 MNaH₂ PO₄ buffer, pH 4.8. Samples were pre-equilibrated in the samebuffer by dialysis. Elution of the column was carried out withsequential buffer applications of 0.01 M, pH 4,8; 0.15 M, pH 4.8; and0.15 M, pH 8.6 NaH₂ PO₄ buffer. Each buffer solution product volume was200 ml.

The 0.15 M NaH₂ PO₄, pH 8.6 eluate was placed on a DEAE-Agarose (Biorad)1.5×30 cm column. The DEAE-Agarose was pre-equilibrated in 0.05 M NaH₂PO₄, pH 4.8 buffer. Elution of the column was carried out withsequential buffer applications of 0.05 M, 0.075 M and 0.5 M NaH₂ PO₄, pH4.8 buffer. Each buffer solution product volume was 200 ml.

The 0.5 M NaH₂ PO₄, pH 4.8 eluent was recycled on the DEAE-Agarose stepgradient for purification of the GCDFP-15 component.

The purified GCDFP-15 thus obtained appears to have an elution volumemolecular size of approximately 70,000 daltons for the concentrated formand 40,000 deltons when it is highly diluted. Thus, this componentappears to be in a polymeric form in gross cystic disease fluid. Thecalculated monomer size obtained by SDS-acrylamide electrophoresis isapproximately 15,000 daltons. Sulfhydryl reducing agents are not neededto produce the monomer formed GCDFP-15 on SDS-acrylamideelectrophoresis.

An attempt to determine the amino acid sequence of GCDFP-15 by directgas phase techniques was unsuccessful since the N-terminal end of theglycoprotein was apparently blocked. Cleavage of GCDFP-15 with cyanogenbromide produced two peptide fragments one of which is blocked. Thecalculated molecular size of one of the peptide fragments from thecyanogen bromide cleavage is approximately 12,500 daltons as determinedby SDS-acrylamide electrophoresis. The partial amino acid sequence ofthis peptide fragment as determined by gas phase techniques is asfollows: ##STR1##

Cyanogen bromide cleavage of purified GCDFP-15 is carried out by firstlyophilizing the protein then adding sufficient distilled water to thelyophilizing material to produce a volume of 1.5 ml. To this solution isadded 75 mM of 1,4-dithiothreitol (DTT), followed by heating at 37° C.for 1 hour. Excess DTT is removed by dialysis against two changes of twoliters water. The protein cleavage reaction is carried out by adding 7.8ml of 97-99% formic acid containing 500 mg cyanogen bromide followed byincubation at 25° C. for 16 hours. Cyanogen bromide is removed bydialyzing the reaction mixture overnight against two changes of 2 literswater. The resulting cyanogen bromide protein cleavage fragments arelyophilized then sufficient distilled water is added to produce a samplevolume of 2.5 ml. The protein cleavage fragments are reduced by adding75 mM DTT followed by incubation at 37° C. for 1 hour. A 250 μl portionis removed from the sample and mixed with 250 μl of N-ethylmopholine. Tothis solution is added 375 μl 5-dimethylamino-1-naphthylenesulfonylchloride (25 mg/ml in dimethyl formamide) followed by vortexing.The dansylation reaction is allowed to proceed at room temperature for 2hours then the dansylated peptides are precipitated by adding 3 ml ofacetone followed by centrifugation for 5 minutes at approximately 1,000RPM. The precipitated peptides are re-washed three times with acetone bythe above procedure. After the final centrifugation spin, the peptideprecipitate is solubilized by addition of 250 μl of 2% SDS and dialyzedovernight against 2% SDS. Lysine, 1 mg, is added to the dansylatedfragments before mixing these peptide fragments with the originalfragments. Sodium thioglycollate, 5 mM, and 10% glycerol is added to thefinal sample just prior to loading 10% of the sample onto polyacrylamidegel electrophoresis slab.

The acrylamide gels utilized in the electrophoresis are prepared in thefollowing manner. A 12% acrylamide gel is prepared by adding 7.5 ml of a40% acrylamide solution (38.8% acrylamide and 1.2%methylene-bis-acrylamide) to 11.25 ml water and 6.25 ml buffer [1.5 Mtris (hydroxymethyl) aminomethane, pH 8.8 and 0.4% SDS]. To thissolution is added 0.08 ml ammonium persulfate and 8 μl ofN,N,N',N'-tetramethylethylenediamine. A 22% acrylamide gel is preparedby adding 13.75 ml of 40% acrylamide solution (38.8% acrylamide and 1.2%methylene-bis-acrylamide) to 5.0 ml water and 6.25 ml buffer [1.5 M tris(hydroxymethyl) aminomethane, pH 8.8 and 0.4% SDS]. To this solution isadded 0.08 ml ammonium persulfate and 8 μlN,N,N',N'-tetramethylethylenediamine. An acrylamide gel gradient slabfrom 12% to 22% is prepared from the above two solutions.

Electrophoresis was carried out by loading 10% of the GCDFP-15 cyanogenbromide peptides cleavage fragments containing the dansylated fragmentsas markers onto the above acrylamide gradient slab. The protein solutionwas placed into 15 wells containing 25 μl each which represented a totalof about 10% of the original cyanogen bromide fragments. Theelectrophoresis buffer contained 47.6 g glycine, 12.12 g of tris(hydroxymethyl) aminomethane and sufficient distilled water to make 4liters. The electrophoresis was performed at 25 V overnight until thedye track moved to approximately 1/2 cm from the bottom of the gel.

After electrophoresis, two distinct fluorescent bands (dansylatedfragments) were observed in the gel by ultraviolet light; both weremarked with India ink. The gels were stained for 1 hour at roomtemperature in staining solution containing 25% isopropanol, 10% aceticacid and 0.25% Coomassie Brilliant Blue dye, then destained in asolution containing 25% methanol and 10% acetic acid.

The 12,500 dalton band in the gel was cut out for elution and sequenceanalysis. A 3.5K membrane was cleaned by washing twice in 1% NaHCO₃ at60° C. then washing once in 1% SDS at 60° C. before use. The cut out gelpiece was placed in the negative side of the elutor with 1 ml of 0.2 MTris acetate pH 8.0, 1% SDS and 1 mg DTT and incubated at roomtemperature for 2 hours before starting the elution. After adding 0.05 MTris acetate pH 7.8 and 0.1% SDS to both sides of the elution chamber,the sample was eluted at 50 v for 2 hours. The outside chamber bufferwas changed and elution then continued at 50 v for 2 days. The elutedsamples were removed and placed in a small elutor to concentrate to 100μl followed by analyses on the amino acid sequenator.

Antibodies selective to GCDFP-15 can be elicited by injecting thepurified component into a suitable animal host. Suitable animal hostsinclude rabbits, guinea pigs, horses, sheep, goats and the like. Rabbitsrepresent a preferred animal host. The GCDFP-15 can be injected in asuitable fluid vehicle such as incomplete Freund's adjuvant. Multipleimmunizations are usually required in order to achieve desirableantibody levels in the host's serum. Bleeding of the host animalprovides the desired antiserum.

In a specific embodiment, antiserum containing antibodies selective toGCDFP-15 was prepared in rabbits. The rabbits were injected weekly witha 1.0 ml. volume of purified GCDFP-15 at 1.0 mg/ml in 50% incompleteFreund's adjuvant. After six immunizations, rabbits were injectedbiweekly and bled by ear vein prior to each injection. Absorption ofantiserum was carried out by addition of the absorbing antigen directlyto a specific quantity of antiserum followed by incubation at 10° C. for24 hours. The precipitate was removed by centrifugation at 2,000 rpm for20 minutes at 10° C.

Radiolabelled GCDFP-15 is readily prepared from the purified protein byprocedures well known in the art. Suitable radiolabelled derivatives ofGCDFP-15 include ³ H-, ¹⁴ C-, ¹³¹ I- and ¹²⁵ I-. Particularly preferredis the ¹²⁵ I-derivative which is produced by radiolabelling the compoundusing the method of Hunter and Greenwood, Nature, 194, 495 (1962).

A suitable radioimmunoassay for the GCDFP-15 component may be performedin the following manner. Polypropylene tubes are used for the reaction.500 μl of 0.1 M ammonium acetate buffer, pH 6.75, is added to each tube.The GCDFP-15 antigen standard for the radioimmunoassay is a 1:10,000dilution of a 0.5 mg/ml solution of purified GCDFP-15. This standard ismade up in 0.1 M ammonium acetate buffer, pH 6.75, containing 1 mg/mlcrystallized human albumin. For the assay, five antigen inhibition curvetubes (in duplicate) receive 0 μl, 25 μl, 50 μl, 75 μl and 100 μl,respectively, of the GCDFP-15 antigen standard which is equivalent to 0ng, 1.25 ng., 2.5 ng., 3.75 ng. and 5 ng. of the GCDFP-15.

Each of the antigen inhibition curve tubes receives 50 μl of a 80 mg/mlsolution of bovine plasma. Experimental tubes (in duplicate) receive 50μl of plasma sample. All assay tubes receive 100 μl of a 1:2,000dilution of antiserum. The assay tubes are incubated at 10° C. for 18hours followed by the addition to each tube of 100 μl of a 1:10 dilutionof stock ¹²⁵ I-radiolabelled GCDFP-15. The tubes are then incubated atroom temperature for five hours. The reaction is stopped by the additionto each tube of 6.5 ml.of zirconyl phosphate gel (Z-gel) suspension in0.1 M ammonium acetate buffer, pH 6.75 (equivalent to 0.5 ml. of Z-gelpellet). Zirconyl phosphate gel is prepared according to the method ofHansen and Miller, Analytical Biochemistry, 7, 129 (1964).

The tubes are centrifuged at 3,000 rpm for five minutes. The supernatentis decanted and the Z-gel precipitate is counted in a Packard gammascintillation spectrometer with a counting efficiency of approximately60%.

The radiolabelled GCDFP-15 in the presence of 50 μl of bovine plasmasolution (80 mg/ml) binds 30 percent of the counts present to the Z-gel.Addition of 100 μl of the 1:2,000 antibody solution to the assay causes60% of the counts present to be bound to the Z-gel. A four-fold increasein antibody concentration will bind over 90% of the counts to the Z-gel.A standard antigen inhibition curve may be prepared for the assay andthe quantity of inhibition of GCDFP-15 in a sample determined therefrom.

Analysis of 50 individual GCD fluid samples, using the above describedradioimmunoassay, indicated a GCDFP-15 antigen concentration in therange of 1-10 mg/ml in unfractionated GCD fluid. Analysis of salivasamples from 20 different normal patients (12 women and 8 men)demonstrated an antigen range of 10-70 μg/ml of GCDFP-15. Human plasmasamples from 92 normal women were found to have an antigen range of 5-85ng/ml with a mean of 31 ng/ml. A study of 1,000 different patientsincluding 300 patients with breast carcinoma has resulted in findingelevated plasma levels, up to 30,000 ng/ml, only for women withmetastatic breast carcinoma.

Radioimmunoassay analysis of tissue culture supernatant fluids from fourday cultures of explants of human breast carcinoma has demonstrated thatapproximately one third of these cultures release significant quantitiesof the GCDFP-15 protein as seen in Table I.

                  TABLE I                                                         ______________________________________                                        Pathological  GCDFP-15 in ng/ml                                               Sample        1      2      3     4    5    6                                 ______________________________________                                        Fibroadenoma  0      0      0     0    0    0                                 Fibroadenoma  0      0      0     0    0    0                                 Fibroadenoma  0      0      0     0    0    0                                 Fibroadenoma  0      0      0     0    0    0                                 Fibrosis      0      0      0     0    0    0                                 Fibrosis      43     36     79    25   44   25                                Sclerosing Adenosis                                                                         145    145    185   90   160  170                               Gross Cystic Disease                                                                        500    175    80    260  135  160                               Gross Cystic Disease                                                                        2210   2158   1482  2392 1976 1430                              Dysplasia     0      0      0     0    0    0                                 Breast Carcinoma                                                                            225    100    130   200  80   185                               Breast Carcinoma                                                                            1040   1170   2470  1144 1170 1430                              Breast Carcinoma                                                                            180    185    155   115  135  135                               Breast Carcinoma                                                                            90     132    122   74   108  114                               Breast Carcinoma                                                                            0      0      0     0    0    0                                 Breast Carcinoma                                                                            0      0      0     0    0    0                                 Breast Carcinoma                                                                            53     70     61    46   35   36                                Breast Carcinoma                                                                            0      0      0     0    0    0                                 Breast Carcinoma                                                                            20     15     15    28   13   21                                Breast Carcinoma                                                                            0      0      0     0    0    0                                 ______________________________________                                         *Each sample of tissue was divided into approximately 20 mgs of tissue fo     culture in petri dishes containing 1 ml of culture media. Each sample was     cultured in six replicate dishes. The culture media from each specimen wa     removed after four days and analyzed for GCDFP15 content. All tissues wer     extensively rinsed in culture media prior to culturing.                  

Benign breast disease specimen culture demonstrated significant releaseof the GCDFP-15 from gross cystic disease tissue and sclerosing adenosistissue (an epithelial proliferation associated with cystic disease).

While the aforesaid radioimmunoassay procedure for detecting GCDFP-15 inbiological fluids has been described in some detail, the method of thepresent invention is not limited to any specific immunoassay procedure.

Thus, in its broadest aspect, the present method is useful for detectingthe presence of GCDFP-15 in biological fluids using the GCDFP-15specific antibody and labelled GCDFP-15. Such labelled GCDFP-15 includesthe radiolabelled GCDFP-15 compounds hereinbefore described, preferably¹²⁵ I-GCDFP-15, or GCDFP-15 labelled with any other unique anddetectable label such as, for example, an electron spin resonance group(see U.S. Pat. Nos. 3,453,288, 3,481,952 and 3,507,879), or withchromophore groups, fluorophor groups, enzymes, red blood cells, latexparticles and the like, using techniques and procedures well known inthe diagnostic field.

Similarly, other radioimmunoassay procedures other than the Z-gelprocedure specifically described herein may be employed by one skilledin the art to effect assay of the GCDFP-15 component. Thus, both biasedand competitive binding type radioimmunoassay procedures may be employedin the practice of this invention.

The clinical utility of the GCDFP-15 plasma assay is further defined andcompared to CEA as a tumor marker.

The CEA assay was performed with the CEA-Roche reagents as described byHansen et al., Human Pathol., 5,139 (1974).

Plasma samples for analysis on both assays were obtained as single 7 ccK₃ ethylenediamine tetra-acetic acid blood tubes and were processed toplasma within three hours of obtaining the blood sample. The plasmasamples were frozen at -20° C. until analysis. Clinic patients hadplasma samples obtained prior to physical examination and specifictherapy. The entire patient population studies consisted of 92 normalwomen and 768 patients with breast diseases being treated either atColumbia Presbyterian Medical Center or Duke University Medical Center.The patients are categorized as follows: 253 women with benign breastdiseases; 164 women with Columbia Clinical Classification Stage A or Bbreast carcinoma; 288 women under observation after mastectomy forprimary breast carcinoma; included in this group are 107 women from theabove Columbia Clinical Classification Stage A or B breast carcinomapatients on whom blood samples have been obtained postoperatively; 216women with metastatic breast carcinoma; included in this group are 46women from the above postmastectomy observation group in whom metastaticdisease developed; the other 170 metastatic breast carcinoma patientshad developed metastatic disease prior to initiation of CEA and GCDFP-15analysis.

Both the CEA and GCDFP assays have previously been found to beinsensitive for detection of early breast carcinoma (Columbia ClinicalClassification Stage A or B breast carcinoma). The utility of bothassays has thus been assessed relative to metastatic carcinoma. A totalof 288 patients with breast carcinoma have been followed aftermastectomy. Forty-six of the 288 patients have developed metastaticrecurrence of breast carcinoma. A stratification of CEA and GCDFP-15plasma levels for these 46 patients is presented in Table II.

                                      TABLE II                                    __________________________________________________________________________    PATIENTS WHO UNDER OBSERVATION AFTER MASTECTOMY                               DEVELOPED METASTATIC BREAST CARCINOMA*                                        LOCATION                                                                              NUMBER GCDFP-15                                                                             CEA   BOTH                                              OF      OF     >150 ng/ml                                                                           >10 ng/ml                                                                           MARKERS                                           METASTASIS                                                                            PATIENTS                                                                             ONLY   ONLY  ELEVATED                                                                             TOTALS                                     __________________________________________________________________________    SOFT TISSUE                                                                           14     0      3 (21%)                                                                             0       3 (21%)                                   OSSEOUS 17     5 (29%)                                                                              6 (35%)                                                                             0      11 (65%)                                   VISCERAL                                                                              15     3 (20%)                                                                              4 (27%)                                                                             1 (7%)  8 (53%)                                           46     8 (17%)                                                                              13 (28%)                                                                            1 (2%) 22 (48%)                                   __________________________________________________________________________     *ANTIGEN ELEVATIONS DEVELOPED EITHER PRIOR TO OR CONCURRENT WITH THE          DIAGNOSIS OF METASTATIC DISEASE                                          

Of the 46 patients who developed breast carcinoma recurrence, 14developed CEA levels above 10 ng/ml and 9 developed GCDFP-15 plasmalevels above 150 ng/ml. Analysis of these 46 patients relative to thesite of their recurrent disease and CEA or GCDFP-15 plasma levelsdemonstrated the highest percentage detection in patients with osseousmetastasis (65%) and the lowest percentage detection in patients withsoft tissue metastasis (21%). Twenty-two of the 46 patients (48%)developed either abnormal CEA or GCDFP-15 plasma levels; only one of the22 patients had elevated plasma levels of both CEA and GCDFP-15. Thus,utilization of both plasma marker assays as monitors for breastcarcinoma recurrence was more effective than utilization of either assayalone.

The utility of the GCDFP-15 or CEA assay to detect recurrence of breastcarcinoma prior to clinical demonstration of recurrence is illustratedby the following case report where the GCDFP-15 assay demonstratedincreasing abnormal levels of this antigen for approximately one yearprior to clinical detection of recurrence.

Case Report 1, Duke Study Number D-87

This patient, a 68 year old female, underwent left radical mastectomy inApril 1975. Infiltrating duct carcinoma with metastases to eight ofsixteen axillary lymph nodes was found. A right simple mastectomy wasperformed at the same time with no pathology found in the right breastspecimen. The patient was begun on adjuvant chemotherapy of Cytoxan,Methotrexate and 5-Fluorouracil (CMF) in June 1975. This adjuvantchemotherapy was continued for one year. Initiation of CEA and GCDFP-15determinations was Oct. 9, 1975 (Day 0). In November 1976 (Day 397) thepatient developed left hip pain for the first time. A bone scandemonstrated increased tracer activity in the neck of the left femur anda chest x-ray indicated a left pleural effusion and several smallnodules in the left lung.

The GCDFP-15 plasma assay had been progressively increasing above 150ng/ml for almost one year since December 1975 (Day 55) and had risen to6000 ng/ml by November 1976 (Day 397). The patient was treated withDiethylstilbestrol and had an objective remission with completeresolution of the left pleural effusion and left lung nodules. She alsohad marked subjective improvement with resolution of left hip pain.

However, in February 1977 (Day 485) she developed sudden shortness ofbreath and the diagnosis of pulmonary embolus was made clinically andsupported by lung scan. She responded well to heparin anticoagulationand was begun again on Diethylstilbestrol along with Salicylate therapyapproximately three weeks (Day 510) after her pulmonary embolus. Herchest x-ray remains clear and her left leg remains asymptomatic althoughthere has been no change by x-ray in the lytic disease located in theleft femur. The patient's most recent GCDFP-15 plasma level taken inJune of 1977 (Day 590) was 315 ng/ml. The CEA plasma levels in thispatient have constantly been below 5 ng/ml and without significantvariation.

Two hundred and sixteen patients treated for metastatic breast carcinomahave been evaluated with serial plasma assays for both CEA and GCDFP-15.The 216 patients have been stratified for evaluation relative to thelocation of their metastatic disease as best determined by clinical andradiological examination, as seen in Table III.

                                      TABLE III                                   __________________________________________________________________________    PATIENTS UNDER TREATMENT FOR                                                  METASTATIC BREAST CARCINOMA                                                   LOCATION                                                                              NUMBER GCDFP-15                                                                             CEA   BOTH                                              OF      OF     >150 ng/ml                                                                           >10 ng/ml                                                                           MARKERS                                           METASTASIS                                                                            PATIENTS                                                                             ONLY   ONLY  ELEVATED                                                                             TOTALS                                     __________________________________________________________________________    SOFT TISSUE                                                                           73      8 (11%)                                                                             11 (15%)                                                                            0      19 (26%)                                   OSSEOUS 62     18 (29%)                                                                             15 (24%)                                                                            16 (26%)                                                                             49 (79%)                                   VISCERAL                                                                              81     12 (15%)                                                                             18 (22%)                                                                            13 (16%)                                                                             43 (53%)                                   TOTALS  216    38 (18%)                                                                             44 (20%)                                                                            29 (13%)                                                                             111 (51%)                                  __________________________________________________________________________

Elevations in CEA (above 10 ng/ml) and in GCDFP-15 (above 150 ng/ml)have occurred at some point in the disease course of 111 (51%) of the216 patients. Of these 111 patients, 44 (20%) had CEA elevations only,38 (18%) had GCDFP-15 elevations only, and 29 (13%) had elevations ofboth CEA and GCDFP-15. Thus, the CEA and GCDFP-15 plasma antigen markerswere elevated independently of one another in 74% of these patients. Ofthe 62 patients with an osseous location of metastatic disease, 49 (79%)had elevated plasma levels of either CEA or GCDFP-15. Of the 81 patientswith visceral metastasis, 43 (53%) had elevated marker antigen levels,and 19 (26%) of the 73 patients with soft tissue metastasis had elevatedvalues.

Of the 29 patients with both CEA abd GCDFP-15 plasma marker elevations,several had a much higher concentration of one antigen relative to theother. It has been observed that the more elevated plasma antigen isusually the best marker to follow as an indicator of therapeuticefficacy. An example case follows.

Case Report 2, Duke Study Number D-601

This patient, a 60 year old female, presented with a lump in the leftbreast of "six months" duration. Breast carcinoma fixed to the chestwall was found associated with large firm axillary lymph nodes. Thepatient underwent a left mastectomy in September 1975. Estrogen receptoranalysis of the carcinoma specimen was positive. By January 1976,supraclavicular lymph node metastases were evident. Initiation of CEAand GCDFP-15 determinations was February 10, 1977 (Day 0). The patientwas started on Aminoglutethimide therapy Feb. 13, 1977 (Day 3). Bone andliver scans at this time were negative. A left pleural effusion waspresent on chest x-ray, however, no discrete pulmonary nodules were seenand no osseous metastases were present. The patient's CEA was 7.0 ng/mland the GCDFP-15 was 2250 ng/ml.

By March 1977 (Day 42) an increase in the left pleural effusion hadoccurred. The CEA had risen to 8.6 ng/ml and the GCDFP-15 had risen to3,350 ng/ml. The patient was discontinued from Aminoglutethimide andadmitted to the hospital for treatment of the left pleural effusion. Byapril 1977 (Day 60) a malignant pericardial effusion was documented. TheCEA was 9.8 ng/ml and the GCDFP-15 was 4,900 ng/ml. Two weeks later thepatient died from metastatic disease. The CEA two days before death (Day72) was 10.5 ng/ml, the GCDFP-15 was 7,500 ng/ml.

Fifty-five of the 216 patients with metastatic breast carcinoma hadestrogen receptor analysis performed on a biopsy specimen of theirmetastasis. These 55 patients have been analyzed relative to positive(>3 fm/mg estrogen receptor) or negative estrogen receptor content andwhether they have developed either abnormal CEA or GCDFP-15 plasmalevels. No association was seen for either CEA or GCDFP-15 plasma levelalterations with estrogen receptor content of the breast carcinomatissue.

Fifty-seven of the 216 patients with metastatic breast carcinoma havebeen followed with serial CEA and GCDFP-15 plasma level determinationsto within two months of their death from metastatic disease. Analysis ofthese 57 patients relative to development of abnormal CEA or GCDFP-15plasma levels indicated 63% developed CEA levels above 10 ng/ml and 45%developed GCDFP-15 plasma levels above 150 ng/ml. These two percentagefigures are thus representative of the total percentage detection ofabnormal CEA or GCDFP-15 levels which would occur during the course ofmetastatic disease.

An age distribution analysis for the 216 patients with metastatic breastcarcinoma relative to the percentage of patients within each age groupwho had abnormal plasma levels of either CEA or GCDFP-15 was made. Thepercentage of abnormal plasma levels of CEA was slightly higher in thelower age groups whereas the percentage of abnormal plasma levels ofGCDFP-15 was slightly higher in the upper age groups. The total rangewas from 31 to 80 years with detection of both antigens in all agegroups.

As with the CEA assay, the serial measurement of GCDFP-15 levels inpatients with metastatic breast carcinoma appeared to be useful inmonitoring the efficacy of therapeutic regimens. For hormonal therapy,alterations in the GCDFP-15 plasma levels have accurately reflectedclinical responsiveness with increasing levels indicative of progressionand decreasing levels indicative of regression. Patients who show adecrease in the GCDFP-15 plasma level demonstrated objective diseaseregression or stabilization, whereas those patients with increasingplasma levels demonstrated disease progression.

Utilization of both the CEA and GCDFP-15 assays allowed detection of 22(48%) of the 46 breast carcinoma recurrences in 288 patients underobservation after mastectomy. The two assays were found to be almostcompletely independent of one another in this patient group, with onlyone of the 22 detected patients having both marker proteins elevated.

Thus, the two plasma marker proteins, CEA and GCDFP-15, both appear tobe useful as monitors for the detection of metastatic breast carcinomaand for assessing the effectiveness of therapeutic regimens in patientswith metastatic disease.

In a further aspect of the present invention GCDFP-15 levels inbiological fluids is assayed using a double antibody radioimmunoassaywhere the second antibody is supported on an insoluble support material.The second antibody is elicited in a host animal of a different speciesthan that in which the first antibody was produced. This aspect isexemplified in a test performed on amniotic fluid to determine thematurity of the fetus and the possible risk of respiratory distresssyndrome in the delivered baby.

One hundred and eleven amniotic fluid samples were obtained byamniocentesis from pregnant women with gestational age fetuses of 16 to42 weeks. The 111 amniotic fluid samples have been analyzed for GCDFP-15content in the following manner.

The amniotic fluid samples were centrifuged at 1000 X G for fiveminutes. The supernatants were decanted and used for analysis.

Polypropylene tubes are used for the reaction. Five hundred ul of 0.1 Mammonium acetate buffer, pH 6.75, containing 1 mg/ml crystallized humanalbumin is added to each tube. The GCDFP-15 antigen standard for theradioimmunoassay is a 1:1,000 dilution of a 0.5 mg/ml solution ofpurified GCDFP-15. This standard is made up in 0.1 M ammonium acetatebuffer, pH 6.75, containing 1 mg/ml crystallized human albumin. For theassay, five antigen inhibition curve tubes (in duplicate) receive 0 ul,25 ul, 50 ul, 75 ul, and 100 ul, respectively of the GDCFP-15 antigenstandard which is equivalent to 0 ng, 12.5 ng, 25 ng, 37.5 ng, and 50 ngof the GDCFP-15. Experimental tubes (in duplicate) receive 50 ul ofamniotic fluid sample.

All assay tubes simultaneously receive 100 ul of a 1:10 dilution ofstock ¹²⁵ I radiolabelled GCDFP-15 and 100 ul of a 1:2,000 dilution ofantiserum. The addition procedure requires immediate mixing of the added¹²⁵ I radiolabelled GCDFP-15 and added antiserum with the test sample.This is easily accomplished with automatic pipetting devicescommercially available such as the Packard Prias automatic diluter. Theassay tubes are incubated at room temperature for three hours. Thereaction is stopped by addition of a second antibody attached to a solidphase bead. The second antibody is typically goat anti-rabbit IgGattached to Kynar as a 2% solid bead suspension in phosphate bufferedsaline pH 7.0, with 1% bovine albumin. The quantity of second antibodyadded is sufficient to complex between 5 and 10 ug of antibody.Typically 0.5 ml of Kynar suspension is added to each tube. The assaytubes are incubated at room temperature for 10 minutes then centrifugedat 3000 RPM for five minutes. The supernatant is decanted and the secondantibody solid phase pellet counted in a Packard gamma scintillationspectrometer with a counting efficiency of approximately 60%.

The radiolabelled GCDFP-15 in the presence of albumin buffer only hasapproximately 5% of the counts precipitated by the solid phase secondantibody. Addition of 100 ul of the 1:2,000 rabbit anti-GCDFP-15antibody solution to the assay caused 60% of the counts present to beprecipitated by the goat anti-rabbit solid phase second antibody.Addition of cold standard GCDFP-15 up to 50 ng to the assay results in acurvalinear inhibition curve of decreasing amount of counts bound to thesecond antibody solid phase pellet.

With the above assay, levels of GCDFP-15 in amniotic fluid between0-1,000 ng/ml can be determined in approximately four hours time.

The level of GCDFP-15 in the 111 amniotic fluid samples relative tovarious ages of gestation is depicted in Table IV.

                  TABLE IV                                                        ______________________________________                                        LEVEL OF GCDFP-15 IN AMNIOTIC FLUID                                                                 Range of                                                Weeks of  Number of   Level    Mean Level                                     Gestation Specimens   (ng/ml)  (ng/ml)                                        ______________________________________                                         0-24     20          0-20      3                                             25-28      4          85-585   321                                            29-32      7          250-1350 665                                            33-36     41          250-4070 979                                            37-42     39          380-7900 2090                                           ______________________________________                                    

In thirty women amniotic fluid samples were obtained within 72 hours ofdelivery. Only four of the 30 samples had GCDFP-15 levels below 700ng/ml. Two of the thirty women delivered babies which developedrespiratory distress syndrome. The amniotic fluid samples from these twowomen had GCDFP-15 levels of 550 and 520 ng/ml. The outer two women withGCDFP-15 plasma levels below 700 delivered babies which did not developany respiratory problems.

Six women have had serial amniotic fluid samples analyzed for GCDFP-15levels. The samples were obtained between 28 and 37 weeks gestation. Allsix women had loglinear increasing levels of GCDFP-15 with increasingage of gestation. The doubling time of GCDFP-15 level in amniotic fluidwas from 10 to 14 days. Extrapolation backward to a 0 level of GCDFP-15gave a gestation age of 22-24 weeks.

The presence of GCDFP-15 in amniotic fluid appears to be a fetal productof third trimester (from 24 weeks to term). The level of GCDFP-15increases in a loglinear fashion towards term. Amniotic fluid levelsbelow 700 ng/ml are correlated with fetal birth which is associated withan increased risk of respiratory distress syndrome.

Another biological fluid other than plasma in which it is useful toanalyze for GCDFP-15 content is urine from metastatic breast carcinomapatients. One hundred and four of the 216 patients with metastaticbreast carcinoma have had urine sample analysis for GCDFP-15 level. Theurine analysis has been carried out methodologically in an identicalmanner to the amniotic fluid analysis. Sixty-eight of the 104 patientshad plasma GCDFP-15 levels below 150 ng/ml, and all 68 patients hadurine levels of GCDFP-15 below 150 ng/mg urine creatinine. Thirty-six ofthe metastatic breast carcinoma patients had plasma GCDFP-15 levelsabout 150 ng/ml (range from 150 ng to 30,000 ng/ml). Twenty-two of these36 patients had urine GCDFP-15 levels above 150 ng/mg urine creatinine(range 150 ng to 20,000 ng/mg urine creatinine).

Urine analysis of 94 patients after operation for primary breastcarcinoma who were without clinical evidence of recurrence and who hadGCDFP-15 plasma levels below 150 ng/ml found all 94 patients had urineGCDFP-15 levels below 150 ng/mg urine creatinine. Serial urine sampleanalysis on 10 normal women has demonstrated no urine levels of GCDFP-15above 150 ng/mg urine creatinine.

A fairly close correlation exists between the plasma GCDFP-15 level andthe urine GCDFP-15 level. The 15 patients which had plasma GCDFP-15levels above 1000 ng/ml also had GCDFP-15 urine levels above 150 ng/mgurine creatinine. An example case of correlation between plasma andurine GCDFP-15 levels follows:

Duke Study Number D-115

This fifty-four year old patient underwent a mastectomy for breastcarcinoma in 1973. In July 1975 metastasis was demonstrated in themandible. The patient was treated with Cytoxan, Methotrexate and5-Fluorouracil until March 1976 when the patient developed significantleukopenia and therapy was changed to Halotestin. The patient's initialGCDFP-15 plasma level taken in October 1975 was 60 ng/ml. By November1976 the plasma level was 190 ng/ml. Advancement of osseous metastasiswas noted at this time and the patient was restarted on CMF chemotherapywith continuation of Halotestin therapy. Four months later in February1977 the GCDFP-15 plasma level was 1060 ng/ml and chemical therapy wasdiscontinued. The patient received six weeks of radiation therapy to thelow back and pelvic area for osseous metastases. In April 1977 thepatient was initiated on Megace. Her GCDFP-15 plasma level was 1320ng/ml. At this time the first GCDFP-15 urine level was obtained and was270 ng/mg urine creatinine. Six weeks later the plasma GCDFP- 15 levelhad risen to 2460 ng/ml and the urine GCDFP-15 level had risen to 410ng/mg urine creatinine. The patient was discontinued from Megace andreceived a second course of radiation therapy to the lower half of herbody. At the end of radiation therapy in July 1977 her plasma GCDFP-15level was 4000 ng/ml and her urine GCDFP-15 was 1000 ng/mg urinecreatinine. She was placed on Prednisone oral therapy and Cytoxan,Methotrexate and 5-Fluorouracil I.V. therapy. Two weeks later her plasmaGCDFP-15 level had fallen to 2640 ng/ml and the urine GCDFP-15 level to650 ng/mg urine creatinine. Her most recent GCDFP-15 plasma level inSeptember 1977 was 2,100 ng/ml and the urine GCDFP-15 level was 520ng/mg urine creatinine.

Preparation of the goat anti-rabbit IgG second antibody supported on theKynar support material can be accomplished as follows. The startingmaterial is unsintered Kynar (vinylidene fluoride) resin powder, grade301 F, Pennwalt Corp. The powder is dispersed in isopropanol(2-propanol) in the proportions of 50 grams Kynar in 1000 ml ofisopropanol. The suspension is then homogenized by a Brinkmann Polytrolfor 5 minutes at a pulse-frequency of 4000 c.p.s. The Kynar-isopropanolmixture is then transferred to a cylinder containing ten liters ofsaline and stirred until dispersed. The Kynar is then allowed to settleout and most of the supernatant is decanted. After two water washes, theKynar is resuspended in phosphate buffered saline (PBS) (pH 7.0) withmerthiolate (0.01%) to yield a 2% Kynar concentration. The Kynar is nowin the activated state and able to accept protein. Serum from goatsimmunized against rabbit IgG was pooled and found to contain 13.72 mg/mlprecipitable antibody protein. While the isopropanol activated Kynar isstirring, 0.25 to 1 ml of undiluted antiserum per gram of Kynar isadded. The mixture is then homogenized again by the polystron for 5minutes at the same pulse-frequency as before. The suspension is thencontinuously stirred at room temperature for a minimum of 6 hoursfollowed by stirring at 4° C. for a minimum of 12 hours. The suspensionis now ready to be washed. This is accomplished by centrifugation at1500 x g for 10 minutes followed by resuspension in PBS (pH 7.0). Thisprocess is repeated once more and the final material resuspended in PBS(pH 7.0) to 20 grams of Kynar per 1000 ml of buffer. The mixture isagain stirred and 0.05 to 0.25 ml of bovine serum albumin per gram ofKynar added. Homogenization with the Polytron at 4000 c.p.s. for 5minutes is the final step in this procedure.

Additional disclosure concerning the preparation and use of Kynar as asolid support for protein materials is found in U.S. Pat. No. 3,843,443.A more general description of second antibodies supported on insolublesupports in provided in U.S. Pat. No. 4,048,298.

I claim:
 1. A method useful in determining the maturity of a fetus whichmethod comprises determining the concentration of the glycoprotein grosscystic disease fluid protein-15 (GCDFP-15) in an amniotic fluid sampleby conducting an immunoassay for said glycoprotein in said amnioticsample utilizing a GCDFP-15 selective antibody and GCDFP-15 labelledwith a unique and detectable label to produce an antigen-antibodyreaction product, separating said product from the said reagents anddetermining the concentration of GCDFP-15 in the amniotic fluid sampleby detecting the quantitative value of said label in either saidreaction product or said reagents and comparing said label value to astandard curve whereby the concentration of GCDFP-15 in said amnioticfluid is directly correlatable to the maturity of said fetus after about24 weeks of gestation wherein said GCDFP-15 is characterized asfollows:(a) a glycoprotein having a calculated monomer size of about15,000 daltons as determined by sodium dodecyl sulfate acrylamide gelanalysis; and (b) immunologically not identical to any components ofplasma as determined by Ouchterlony analysis; and (c) immunologicalcross identity with a component of human milk and human saliva; and (d)cleavage with cyanogen bromide provides two peptides one of which isblocked, and one of the peptides has a molecular weight of about 12,500daltons as determined by sodium dodecyl sulfate acrylamide gel analysisand having the following partial sequence of amino acids: ##STR2## 2.The method of claim 1 wherein said amniotic fluid sample is obtainednear the time of delivery and the concentration of GCDFP-15 found insaid sample is compared to a pre-determined cut-off level whereby aconcentration below said cut-off level is indicative of potential fetalimmaturity with associated respiratory distress syndrome in thedelivered baby.
 3. The method of claim 1 wherein said immunoassay is aradioimmunoassay and said labelled GCDFP-15 is GCDFP-15 labelled with aradioactive isotope.
 4. The method of claim 3 wherein said radioactiveisotope is ¹²⁵ I.
 5. The method of claim 3 wherein the antigen-antibodyreaction is precipitated, the counts of the precipitate detected and theamount of GCDFP-15 in the amniotic fluid sample is determined from astandard inhibition curve.